Transforming growth factor beta1 (TGF-beta) is widely acknowledged as a potent inhibitor of human and mouse mammary epithelial proliferation. We have localized its activity in the mouse mammary gland and characterized the proliferative phenotype of the Tgfbeta1 heterozygote mouse: both are regulated by the ovarian hormones estrogen and progesterone. It is well known that estrogen signaling through estrogen receptor ? (ER) plays a central role in mammary epithelial cell proliferation. Several recent studies have shown that the majority of estrogen receptor-positive (ER+) cells do not proliferate, but are required to regulate proliferation in ER-negative (ER-) cells via paracrine mechanisms. The small subpopulation (approximately 1% in human and mouse breast) of ER+ cells that maintain proliferative potential has been postulated to be early progenitor cells that could be the origin of ER+ breast cancer. We found that nearly all of the ER+ cells in mouse mammary gland at estrus co-localize with intense TGF-beta activity, consistent with their non-proliferative status. However, proliferation of ER+ cells is increased more than 2-fold in Tgfbeta1 heterozygote mammary gland, which furthermore resulted in a 30% increase in the total number of ER+ cells compared to wildtype mammary epithelium. Based on these observations we hypothesize that TGF-beta actively restrains proliferation of mammary epithelial cells and, in particular, regulates the subpopulation of ER+ cells that are speculated to represent lineage-restricted progenitor. To the extent that stem cells/progenitors are targets for carcinogenesis, we further hypothesize that disruption of TGF-beta action is involved in the genesis of ER+ breast cancer. Our overall goal, therefore, is to define the functional relationship between TGF-beta1 and the population of ER+ replicative cells as a function of age. Three aims are proposed: 1: To determine the frequency of ER+ cells as a function of TGF-beta1 level, localization and activity. 2: To analyze TGF-beta effects on the replicative potential of presumptive mammary stem cells. 3: To determine whether TGF-beta regulation of the ER+ subpopulation contributes to progression and/or features of preneoplastic lesions.